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Method for synthesizing secondary metabolites of saccharopolyspora erythraea by controlling gene dasR and application of gene dasR

A technology for Saccharopolyspora rubrum and secondary metabolites, which can be applied in the field of genetic engineering and can solve problems such as low efficiency, time-consuming and labor-intensive efficiency, and lack of genome sequence information.

Inactive Publication Date: 2015-11-11
EAST CHINA UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] In the early days, due to the lack of genome sequence information due to backward technology, the transformation of some antibiotic-producing actinomycetes was mainly based on multiple rounds of random mutagenesis of wild bacteria, and then a large number of high-yielding mutant strains were obtained through screening. Operational strain optimization methods are time-consuming, labor-intensive and often inefficient

Method used

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  • Method for synthesizing secondary metabolites of saccharopolyspora erythraea by controlling gene dasR and application of gene dasR
  • Method for synthesizing secondary metabolites of saccharopolyspora erythraea by controlling gene dasR and application of gene dasR
  • Method for synthesizing secondary metabolites of saccharopolyspora erythraea by controlling gene dasR and application of gene dasR

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Embodiment 1

[0031] Construction of dasR Gene Knockout Engineering Bacteria Using Double Crossover Homologous Recombination Gene Knockout Technology

[0032] 1.1 Construction of dasR gene knockout plasmid

[0033] The pUC18-tsr plasmid is a 1.36 kb thiostrepton resistance gene (tsr) inserted between the BamHI and SmaI restriction sites of the pUC18 plasmid. In order to knock out the dasR gene in the Saccharopolyspora rubrum genome, dasR-up-fw / rev and dasR-dw-fw / rev were used as primers respectively, and the Saccharopolyspora rubrum genome was used as a template to amplify the dasR gene by PCR. Homology arm DNA fragments of about 1.5kb each in the upstream and downstream.

[0034] The primers used to amplify the DNA fragment of the upper homology arm are:

[0035] dasR-up-fw:5'-AACTGCAGGTCTCGGCCAGCTTCAGGG-3'

[0036] dasR-up-rev:5'-GCGGGATCCCGTTCCTCCCTACCACCGCAA-3'

[0037] The primers used to amplify the DNA fragment of the lower homology arm are:

[0038] dasR-dw-fw:5'-TATGGTACCTACAA...

Embodiment 2

[0063] Determination of the Growth of DasR Gene Knockout Engineering Bacteria and the Production of Secondary Metabolites

[0064] 1.1 Determination of bacterial growth

[0065] Saccharopolyspora rubrum was activated in TSB, and R2YE plates were prepared and cellophane was laid on the surface, and wild-type strains and △dasR strains were inoculated respectively. Cultivate in a constant temperature incubator at 30°C, remove the cellophane and collect the bacteria at 24h, 36h, 48h, 60h, 72h, 96h, 108h and 120h respectively, and collect 3 plates of bacteria for each strain at each time point . Collect the thalli of 3 plates respectively in the 1.5ml centrifuge tube that has been weighed (referred to as the initial amount), and then dry it overnight (12h) in a 50°C oven to make it completely dry, and then weigh it (weighed). is the final amount), subtract the initial amount from the final amount to obtain the dry weight of the thalline (in milligrams).

[0066] The result is as...

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Abstract

The invention relates to a method for synthesizing secondary metabolites from an erythrocin-producing strain, namely saccharopolyspora erythraea under the influence of a dasR gene and an application of the dasR gene. The method comprises the following step: knocking out the dasR gene in a saccharopolyspora erythraea genome to obtain mutant engineering bacteria, wherein the nucleotide sequence of the dasR gene is a sequence in a sequence table. The invention also relates to a mutant strain obtained by knocking out of the dasR gene or an application of the mutant strain in influencing the yield of the secondary metabolites, wherein the secondary metabolites are erythrocin and reddish brown pigment specifically. Compared with a wild type strain in which the dasR gene is not knocked out, the engineering strain in which the dasR gene is knocked out has the characteristic that the amount of produced erythrocin and the pigment is reduced, which indicates that the gene is positively correlated to the production of the secondary metabolite.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and mainly relates to a method for synthesizing secondary metabolites influenced by dasR gene on erythromycin-producing bacterium Saccharopolyspora rubrum and its application. Background technique [0002] Saccharopolysporaerythraea is a Gram-positive filamentous bacterium originally thought to belong to the genus Streptomyces. Belonging to the genus Saccharopolyspora. [0003] Saccharopolyspora rubrum is the main producer of erythromycin, which is mainly used to kill Gram-positive pathogens. This macrolide broad-spectrum antibiotic has been more than 60 years since it was first discovered by J.M.McGurie. However, erythromycin is still widely used in medicine because of its high therapeutic effect and safety. Since then, people have modified the molecular structure of erythromycin A and evolved a variety of more effective derivatives. These semi-synthetic second-generation derivat...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12P19/62C12P1/04C12R1/01
Inventor 叶邦策廖成衡姚荔丽
Owner EAST CHINA UNIV OF SCI & TECH
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